MicroRNAs and hepatitis C virus: Toward the end of miR-122 supremacy

<p>Abstract</p> <p>The most common etiologic agents causing chronic hepatitis are hepatitis C and B viruses (HCV and HBV, respectively). Chronic infection caused by HCV is considered one of the major causative agents of liver cirrhosis and hepatocellular carcinoma worldwide. In combination with the increasing rate of new HCV infections, the lack of a current vaccine and/or an effective treatment for this virus continues to be a major public health challenge. The development of new treatments requires a better understanding of the virus and its interaction with the different components of the host cell. MicroRNAs (miRNAs) are small non-coding RNAs functioning as negative regulators of gene expression and represent an interesting lead to study HCV infection and to identify new therapeutic targets. Until now, microRNA-122 (miR-122) and its implication in HCV infection have been the focus of different published studies and reviews. Here we will review recent advances in the relationship between HCV infection and miRNAs, showing that some of them emerge in publications as challengers against the supremacy of miR-122.</p

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage*

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of αENaC using the serine protease subtilisin. Trimeric channels formed with αFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ∼10-fold decrease in amiloride-sensitive whole cell conductance as compared with αWT (2.2 versus 21.2 microsiemens (μS)). Both αWT and αFM were activated to similar levels by subtilisin cleavage. Channels formed with αFD, a construct that deleted the segment between the two furin sites (Δ175–204), exhibited an intermediate conductance of 13.2 μS. More importantly, αFD retained the ability to be activated by subtilisin to 108.8 ± 20.9 μS, a level not significantly different from that of subtilisin activated αWT (125.6 ± 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of α was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with αLD, a construct that extended the deleted sequence of αFD by 17 amino acids (Δ175–221). Channels with αLD were uncleaved, exhibited low baseline activity (4.1 μS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex

Correction: Permissivity of Primary Human Hepatocytes and Different Hepatoma Cell Lines to Cell Culture Adapted Hepatitis C Virus.

[This corrects the article DOI: 10.1371/journal.pone.0070809.]

Permissivity of primary human hepatocytes and different hepatoma cell lines to cell culture adapted hepatitis C virus.

Significant progress has been made in Hepatitis C virus (HCV) culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells' permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B) and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates

Identification and characterization of potential adaptive mutations.

<p>(<b>A</b>) Positions of conserved mutations found in the adapted virus on JFH1 open reading frame schematic diagram. The originally introduced amino acid changes F172C and P173S in Core and A4 MAb epitope in E1 are indicated in underlined gray type. Mutations identified at the end of the selection are indicated in black type. (<b>B</b>, <b>C</b>, <b>D</b>) Effect of the potential adaptive mutations on viral genome replication, infectious virus production and HCVcc assembly/secretion. HuH-7-RFP-NLS-IPS cells were transfected with JFH1-CS-A4-RLuc RNA (WT) or mutated HCV genomes (I599V, R1373Q, M1611T, S2364P, C2441S, R2523K, R1373Q/C2441S (DM for double mutant) or R1373Q/M1611T/C2441S (TM for triple mutant)). An assembly-deficient virus (ΔE1E2) and a replication-defective virus (GND) were used as controls. (<b>B</b>) Replication was assessed at 4, 24 and 48 h by measuring <i>Renilla</i> Luciferase activities in transfected cells. Results are expressed as relative light units (RLU) normalized at 4 h and are reported as the means ± S.D. of two independent experiments. (<b>C</b>) The supernatant of transfected cells were recovered at 24 and 48 h and incubated for 3 h with naive HuH-7-RFP-NLS-IPS cells. Luciferase assays were performed on infected cells at 72 h post-infection. Results are expressed as RLU and are reported as the means ± S.D. of two independent experiments. (<b>D</b>) HCV core release was quantified in the supernatants recovered 48 h post-transfection. Results are expressed as Core fmol/L and are reported as the means ± S.D. of two independent experiments.</p

Viral entry of cell culture adapted HCV.

<p>(<b>A</b>, <b>B</b>, <b>C</b>) Neutralization of cell culture adapted HCV with 3/11 and JS-81 MAbs. HuH-7-RFP-NLS-IPS cells were infected with i0 or i24 in the absence (Mock) or the presence of 3/11 anti-E2 or JS-81 anti-CD81 MAbs, at the indicated concentration. (<b>A</b>) Images taken 48 h after infection with i24 are representative of three independent experiments. (<b>B</b>, <b>C</b>) Intracellular HCV RNA was quantified 48 h after infection. Results are expressed as percentages of infectivity relative to infectivity in the absence of antibodies and are reported as the means ± S.D. of two independent experiments. (<b>D</b>) Cell-to-cell transmission of cell culture adapted HCV. Naive HuH-7-RFP-NLS-IPS cells (acceptor cells) were seeded with HuH-7-EGFP-IPS cells, infected with either i0 or i24 (donor cells). Cultures were treated with 50 µg/mL of the 3/11 anti-E2 neutralizing MAb to prevent cell-free infection. The results are expressed as the mean number of HCV infected acceptor cells/focus ± S.D., determined in 140 separate foci, 24 h post-seeding.</p